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Journal of Virology, October 2000, p. 8953-8965, Vol. 74, No. 19
Department of Microbiology and Immunology,
Stanford University School of Medicine, Stanford, California
94305,1 and Department of Molecular,
Cellular and Developmental Biology, University of Colorado,
Boulder, Colorado 803092
Received 2 May 2000/Accepted 14 July 2000
All positive-strand RNA viruses of eukaryotes studied assemble RNA
replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with
endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of
Golgi-derived or endosomal membranes in buoyant density gradients,
although changes in ionic strength affected ER and virus-induced
vesicles, but not other cellular organelles, similarly. When expressed
in isolation, two viral proteins of the poliovirus RNA
replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B
and 2C proteins, membranes identical in buoyant density to those
observed during poliovirus infection were formed. When coexpressed with
2BC, viral protein 3A was quantitatively incorporated into these
fractions, and the membranes formed were ultrastructurally similar to
those in poliovirus-infected cells. These data argue that
poliovirus-induced vesicles derive from the ER by the action of viral
proteins 2BC and 3A by a mechanism that excludes resident host
proteins. The double-membraned morphology, cytosolic content, and
apparent ER origin of poliovirus-induced membranes are all consistent
with an autophagic origin for these membranes.
Infection with positive-strand RNA
viruses results in a range of membrane morphologies, many of which
involve complex membrane rearrangements. Cells infected with poliovirus
and other picornaviruses, for example, accumulate large quantities
of membranous vesicles 150 to 400 nm in diameter (Fig.
1) (5, 12). Most of
these vesicles are surrounded by double lipid bilayers (Fig. 1B
and C) (41), precluding a simple budding mechanism.
Instead, the presence of a double membrane suggests a wrapping
mechanism for vesicle formation akin to the process of cellular
autophagy, as suggested previously (12, 41). For all
positive-strand RNA viruses studied to date, the RNA synthesis
machinery is associated with the cytoplasmic surface of these
cytoplasmic membranes, and many of the proteins required for viral RNA
synthesis are membrane associated when expressed in isolation.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Remodeling the Endoplasmic Reticulum by Poliovirus Infection and
by Individual Viral Proteins: an Autophagy-Like Origin for
Virus-Induced Vesicles
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Electron micrographs of COS-1 cells preserved by
high-pressure freezing show the ultrastructure of uninfected cells and
cells infected with poliovirus for 4 h at 37°C. (A) Uninfected
cell. N, nucleus; G, Golgi. Bar = 1 µm. (B) Infected cell;
bar = 1 µm. (C) Infected cell; bar = 0.2 µm. (D)
Immunostaining of infected cell to identify poliovirus 2C epitopes
using 15-nm gold particles conjugated to secondary antibodies. Arrows
indicate double-membraned vesicles. Bar = 0.3 µm.
Studies of several different positive-strand RNA viruses, including costaining and coisolation of viral and cellular proteins known to be residents of individual organelles, have diversely implicated the endoplasmic reticulum (ER), trans-Golgi endosomes, or lysosomes as likely sources for virally induced membranes. For example, Semliki Forest virus has long been thought to replicate on cellular endosomes modified during viral infection (19), and cowpea mosaic virus replication complexes colocalize with ER marker proteins (9). For poliovirus-infected cells, although images consistent with the budding of these vesicles from ER membranes have been reported (5), immunoisolation of the vesicles showed the presence of protein markers from the entire secretory apparatus, including ER, trans-Golgi, and lysosomal markers (41). It was concluded that the poliovirus-induced vesicles either derive from a pooled compartment or acquire many of their cellular markers after their formation (41).
To identify viral proteins which can, in isolation, cause membrane rearrangements, individual proteins from several positive-strand RNA viruses have been expressed in a variety of cell types. For poliovirus, the viral 2C protein, a 329-amino-acid membrane-associated protein with RNA-dependent ATPase activity (31, 38), is required for RNA replication and was shown to cause both membrane vesiculation and the formation of multilamellar structures when expressed in isolation (1, 10). When the 426-amino-acid precursor protein, 2BC, was expressed in human cells, a greater amount of vesiculation was observed, which was morphologically more consistent with the pattern seen in poliovirus-infected cells. The more authentic ultrastructural changes were seen only when 2B and 2C were covalently linked (1, 10, 48).
An 87-amino-acid protein, 3A, when expressed in isolation, associates with membranes of the ER, as shown by immunoelectron microscopy (13) and by coimmunofluorescence (14). Near the C terminus of the 3A coding region is a hydrophobic region whose integrity is required for membrane association in vitro (49) and is presumed to anchor 3A and 3AB to cellular membranes during poliovirus RNA synthesis. Membrane-associated 3AB binds directly to the poliovirus RNA-dependent RNA polymerase (22), stimulating protease activity of the polymerase precursor 3CD (25), and thus is thought to anchor 3D polymerase in the RNA replication complexes. Either the 22-amino-acid 3B or its precursor 3AB serves as the primer for RNA synthesis (33). Although 3A protein expression causes the inhibition of ER-to-Golgi protein traffic (14) and dramatically alters ER ultrastructure (13), these morphological changes do not in themselves resemble those observed in poliovirus-infected cells.
Autophagic vacuoles form in normal cells in response to nitrogen or
amino acid starvation. Membranes thought to be derived from the ER
(16) wrap around small pools of cytoplasm, forming immature
autophagic vacuoles. The lumen of these double-membraned, immature
autophagic vacuoles contains material morphologically indistinguishable
from cytoplasm, sometimes including intact mitochondria or other small
organelles. The evidence that autophagic vacuoles form from the ER has
been predominately ultrastructural (16). Biochemical
identification of the origin of the limiting membranes in these
structures has proven difficult, possibly because these membranes are
relatively depleted for protein markers (37, 45). However,
those proteins present in immature autophagic vacuoles include the ER
lumenal proteins calreticulin, protein disulfide isomerase (PDI), ER60
protease, and BiP (51). As the double-membraned autophagic
vacuoles mature, they acquire from vesicles in the endosomal and
lysosomal pathway protein markers and lumenal contents such as
-hexaminidase (17). The degradation of the inner lipid bilayer marks the transition to single-membraned, mature autophagic vacuoles (17). Poliovirus-induced vesicles resemble immature autophagic vacuoles in that they are frequently bounded by double membranes, bear markers from the late as well as early secretory pathway, and contain lumenal material indistinguishable from cytoplasm, sometimes including cytoplasmic organelles such as mitochondria (12, 41).
Here, we provide biochemical and ultrastructural evidence for the effects of expression of individual viral proteins on host cytoplasmic membranes. Density gradient centrifugation was used to identify the host membranes with which the individual viral proteins 2C, 2BC, and 3A associate when expressed in isolation and during poliovirus infection. Corresponding ultrastructural studies of infected cells and cells transfected with individual viral proteins were performed using high-pressure freezing and cryosubstitution to preserve membrane morphology (reviewed in reference 11). The combined actions of 2BC and 3A were found to mimic both the biochemical and ultrastructural alterations in poliovirus-infected cells, forming structures consistent with an autophagic mechanism for the formation of the virus-induced vesicles from the ER.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell culture and virus infection. COS-1 cells were cultured as monolayers in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 5 mM glutamine, and 30,000 U of penicillin-streptomycin (Life Technologies, Inc., Gaithersburg, Md.) per liter at 37°C in a 5% CO2 incubator. Wild-type poliovirus type 1 Mahoney was used to infect cells as previously described (30). All infections were performed at a multiplicity of 50 PFU per cell. Virus titers were determined by plaque assays on COS-1 cells.
Plasmids and DNA transfections. The pLINK-based DNA plasmids used in these experiments have been previously described (14). Briefly, these constructs, which are under the transcriptional control of the simian virus 40 late promoter, contain an internal ribosomal entry site followed by the coding sequence for the vesicular stomatitis G protein (VSV-G). In the parental pLINK plasmid, only VSV-G is produced. No effect of VSV-G expression from pLINK was observed in either ultrastructural or gradient fractionation experiments (data not shown). The dicistronic pLINK derivatives used here encode poliovirus protein 2B, 2C, 2BC, or 3A in the first cistron as well as VSV-G in the second cistron. DNA transfections were performed using Lipofectamine or Lipofectamine Plus reagents as described by the manufacturer (Life Technologies). Twelve micrograms of each plasmid DNA was used for transfection of each 150-mm-diameter plate of 4 × 106 COS-1 cells. Following a 48-h incubation period, cells were harvested and either used in biochemical fractionation experiments or prepared for electron microscopic analysis. The 48-h incubation time point yielded ultrastructural results similar to results of 24-h incubations for those proteins tested (data not shown), but expression levels of the viral proteins were higher at the later time point, which facilitated biochemical analysis.
Biochemical fractionation. Cells were infected with poliovirus at 37°C for 4 h unless otherwise specified or transfected for 48 h at 37°C with the pLINK-based DNA plasmids. Each sample of 1.5 × 108 infected, transfected, or control cells was scraped into phosphate-buffered saline (PBS), collected by low-speed centrifugation, and resuspended in 1 ml of homogenization buffer (0.25 M sucrose, 5 mM morpholinepropanesulfonic acid [pH 7.4]). The plasma membranes were disrupted by pressure filtration through 14-µm-pore-size polycarbonate track-etch membranes (Osmonics, Livermore, Calif.), a technique useful in preserving the integrity of organelles during cell lysis (46). The low-salt conditions used were found to be optimal for the separation of cytoplasmic organelles from the poliovirus-induced membranes (Fig. 2 and 8) and did not lead to nonspecific aggregation, as evidenced by the successful separations achieved. The filtrate was centrifuged twice for 10 min at 4°C and 1,000 × g to sediment intact cells, nuclei, and large sheets of plasma membranes. The resultant supernatant was loaded onto 20 ml of self-forming Percoll density gradient medium (Pharmacia Biotech, Piscataway, N.J.); the Percoll concentrations indicated for each experiment were achieved by diluting stock isotonic Percoll (9 parts Percoll, 1 part 2.5 M sucrose [vol/vol]) with homogenization buffer. For some experiments, both the homogenization buffer and the Percoll suspension medium were adjusted to 60 mM KCl. The samples were centrifuged in a Beckman type 60 Ti rotor at 25,000 rpm for 23 min at 4°C. Density marker beads (Pharmacia Biotech) were used to calibrate the initial gradients (data not shown).
Following centrifugation, 40 individual 0.5-ml fractions were collected from each sample using a density gradient fractionator (Brandel, Gaithersburg, Md.); each fraction was adjusted to 1 mM phenylmethylsulfonyl fluoride and 0.1% Triton X-100. For direct analysis of lysosome-derived material, equivalent volumes of each fraction were measured for-hexosaminidase activity (36).
Briefly, 200 µl of substrate buffer (50 mM sodium citrate [pH 4.8],
0.1% Triton X-100, 1.7 mg of
p-nitrophenol--N-acetylglucosamide [Sigma, St. Louis, Mo.] per ml) was added to 100 µl of each sample. After incubation for 60 min at 37°C, the reaction was developed by adding 0.9 ml of 0.15 M glycine (pH 10.8). Release of the
p-nitrophenol product was measured by increased absorbance
at 412 nm.
For immunoblot analysis, equivalent volumes of each sample were
subjected to centrifugation at 100,000 × g at 4°C
for 45 min in a Beckman TLA-100 rotor to pellet the Percoll resin. The
proteins in the resultant supernatants were precipitated
(55), resuspended in a minimal volume, and displayed by
glycine-sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
(PAGE) or tricine-SDS-PAGE (40). The proteins were then
transferred to Immobilon-P polyvinylidene fluoride membranes
(Millipore, Bedford, Mass.), which were probed directly using
antibodies generated against specific viral proteins or to resident
marker proteins of cellular organelles. To identify the distribution of
poliovirus proteins, anti-2C monoclonal antibodies, kindly provided by
K. Bienz and D. Egger (University of Basel, Basel, Switzerland), were
used at a dilution of 1:1,500, and anti-3A monoclonal antibodies
(13) were used at a dilution of 1:50. Fractions containing
proteins from the ER were identified either by an anticalnexin
polyclonal serum (Stressgen, Victoria, British Columbia, Canada) at a
dilution of 1:4,000 or by anti-p63 monoclonal antibodies, kindly
provided by H.-P. Hauri (University of Basel). Anti-p115 (7DI)
monoclonal antibodies, kindly provided by G. Waters (Princeton
University) and used at a dilution of 1:20,000, and anti-1,4-galactosyltransferase polyclonal serum, kindly provided by E. Berger (Universität Zurich, Zurich, Switzerland) and used at a
dilution of 1:250, were used to identify Golgi-containing fractions.
Mitochondrial fractions were tracked using anti-mitochondrial HSP70
(mtHSP70) monoclonal antibodies, kindly provided by S. Pierce (Northwestern University), at a dilution of 1:500. Finally, endosomes were traced with anti-Rab9 monoclonal antibodies, kindly provided by S. Pfeffer (Stanford University), at a dilution of 1:100. Anti-mouse and
anti-rabbit secondary antibodies, conjugated to alkaline phosphate (Zymed, South San Francisco, Calif.), were used at 1:4,000. Enhanced chemifluorescence (Amersham, Arlington Heights, Ill.) coupled with
PhosphorImager analyses utilizing ImageQuant (version 4.0) software
(Molecular Dynamics, Sunnyvale, Calif.) was used to generate signals
that were linearly responsive to protein concentrations and to quantify
the relative level of each protein marker. The results are presented as
the percentage of the specific protein found within each fraction; the
total amount of the protein of interest was determined by summing the
enhanced chemifluorescence signals from the 40 gradient fractions.
High-pressure freezing and freeze-substitution.
For
cryofixation and electron microscopy, transfected, infected, or
untreated COS-1 cells from one 100-mm-diameter plate were washed twice
with PBS, removed from the plates by treatment with trypsin, and
collected by centrifugation. The cell pellet was resuspended in a
minimal volume of 0.15 M sucrose in PBS. Aliquots of the cell slurry
were frozen in a Balzers HPM 010 high-pressure freezing apparatus as
described previously (41) and stored in liquid nitrogen. For
observation of cellular morphology, samples were freeze-substituted in
0.1% tannic acid in acetone at 
80°C, rinsed in acetone, then
warmed to 20°C in the presence of 2% osmium tetroxide in acetone
for 16 h, and incubated at 4°C for 4 h. After being rinsed
in acetone at 4°C, samples were embedded in Epon-Araldite resin. Thin
sections were stained with 2% uranyl acetate and lead citrate and then
imaged at 80 kV in a JEOL 100C or Philips CM10 electron microscope.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Poliovirus-induced vesicles do not cofractionate with ER, Golgi,
lysosomal, or mitochondrial protein markers.
Buoyant density
gradients were used to determine the proportion of protein markers from
ER, Golgi, lysosomes, and mitochondria found in
poliovirus-induced vesicles and in subcellular organelles during
poliovirus infection. Separation of cellular components by
centrifugation in polydisperse solutes such as Percoll is based on a
combination of sedimentation rate and isopycnic effects, with the
density of the organelles being the most critical parameter for
separation (43). To fractionate the cytoplasmic organelles in uninfected and in poliovirus-infected COS-1 cells, plasma membranes were disrupted by pressure filtration homogenization (46);
nuclei and intact cells were removed by low-speed centrifugation, and the postnuclear supernatants were fractionated on Percoll gradients. Fractions were collected and assayed either enzymatically or by SDS-PAGE followed by transfer to membranes for Western blot analysis. Membranes associated with poliovirus RNA replication complexes were
identified using antibody to poliovirus protein 2C, known to be
associated with the virus-induced vesicles and with active RNA
replication complexes (7). The poliovirus 2C-containing fractions were narrowly distributed upon fractionation in 20% stock
isotonic Percoll (Fig. 2A), although the
fractions that contained the poliovirus RNA replication complexes
became more disperse at lower Percoll concentrations (Fig. 2B and C).
Thus, the range of buoyant densities displayed by the
poliovirus-induced vesicles is narrow and Percoll gradients can be
efficiently used in their purification.
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-hexosaminidase activity (Fig. 2C) to identify the
lysosome-containing fractions in 9.5% stock isotonic Percoll gradients. The discrete peak of -hexosaminidase activity
observed in uninfected cells was not significantly altered after 4 h of poliovirus infection, and very little overlap was seen between the
-hexosaminidase- and poliovirus 2C-containing fractions.
Mitochondria form an unlikely source for the poliovirus-induced
vesicles, and no disruption of mitochondria has been reported during
poliovirus infection. Therefore, as a representative organelle that
should not be disrupted during poliovirus infection, the fractionation
of mitochondria in 9.5% stock isotonic Percoll gradients was
determined by immunoblot analysis using antibodies generated against
the mtHSP70 (20). In mock- and poliovirus-infected cells, mitochondria formed a discrete peak that cofractionated with
lysosomes (Fig. 2C). The mitochondrial peak was not disrupted by
poliovirus infection, and the majority of the poliovirus 2C-containing
membranes did not contain the GRP75 mitochondrial marker.
Thus, the poliovirus-induced vesicles are either generated de novo,
formed from a subcellular compartment whose resident proteins were not
tested, or generated from one of the tested organelles (ER, Golgi,
lysosomes, or mitochondria) by a mechanism that excludes at least some
of the resident proteins. To test the latter possibility, we sought to
determine whether the characteristic biochemistry and
intracellular morphology of poliovirus-infected cells could be
mimicked by the expression of a subset of the viral proteins and, if
so, to determine their intracellular localization.
Viral proteins 2C and 3A fractionate as resident ER proteins and
alter ER morphology when expressed in isolation.
Previous studies
have shown that the ER membranes of cells that expressed the 3A protein
displayed a characteristic swollen and distended morphology by electron
microscopy (13). Immunoelectron microscopy demonstrated that
3A protein localizes to these swollen membranes (13).
Coimmunofluorescence of the ER marker PDI secretory cargo (
-1
protease inhibitor) whose transport was disrupted by 3A function, and
3A protein showed that the swollen membranes that contain both 3A and
arrested secretory cargo are ER (13, 14). To examine
the distribution of 3A protein in Percoll gradients, COS-1 cells were
transfected with a plasmid that expressed 3A protein from a dicistronic
RNA that also encoded VSV-G (14). Cell extracts were
prepared and fractionated on 12% stock isotonic Percoll gradients, the
concentration found to be optimal for separation of the organelles of
interest. The fractions that contained 3A protein were identified by
immunoblot analysis. The positions of ER, Golgi, lysosomal, and
mitochondrial membranes were also determined, although only a subset of
these distributions is shown for simplicity. In both transfected and
untransfected COS-1 cells, the ER marker calnexin was dispersed over
many fractions; 3A protein in the transfected cells cofractionated with
the ER fractions of the lightest buoyant density (Fig.
3A). The expression of 3A protein did not
perturb the distribution of ER membranes in Percoll gradients compared
to untransfected cells (data not shown); because only 30 to 40% of the
cells were transfected, a subtle disturbance in the transfected cells
may not have been detectable. Electron microscopy was performed on
populations of cells that were transfected with 3A-expressing plasmids.
Approximately 40% of the cells displayed the characteristic morphology
reported previously to be caused by 3A expression (Fig. 3B)
(13).
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Viral protein 2BC in isolation forms membranes of the buoyant
density observed in poliovirus-infected cells.
Previous electron
microscopic studies of cells that express individual poliovirus
proteins have suggested that the expression of poliovirus 2BC protein
mimics the morphology of poliovirus-infected cells (1, 10,
48). To test the effect of 2BC protein expression on the
biochemical distribution of cytoplasmic membranes in Percoll gradients, COS-1 cells were transfected with a DNA plasmid that expressed 2BC and cytoplasmic extracts were fractionated on a 12%
stock isotonic Percoll gradient (Fig.
5A). The virally encoded protease
responsible for cleavage of the 2BC protein was not expressed in the
transfected cells; therefore, only the 48 kDa 2BC protein, and not its
final processing products, was detected by immunoblot analysis (data
not shown). Like the membranes from poliovirus-infected cells, but
unlike cells expressing 3A and 2C, the peak of the 2BC-containing
membranes displayed a lighter buoyant density, centered at fraction 11, than even the lightest of the ER-containing fractions, centered at
fraction 13 (Fig. 5A). Therefore, 2BC expression in isolation was
sufficient to generate membranes of buoyant densities similar to those
observed in poliovirus-infected cells.
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2BC expression causes 3A protein to change its localization from
the ER to a fraction with a lower buoyant density.
The effect of
coexpression of 2BC and 3A was tested in an attempt to mimic more
closely the biochemistry and ultrastructure of poliovirus-infected
cells. COS-1 cells were cotransfected with plasmids that expressed 2BC
and 3A, and cytoplasmic extracts were displayed on 12% stock isotonic
Percoll gradients (Fig. 6A). As in Fig.
5A with 2BC alone, 2BC and 3A coexpression led to the formation of a
peak of lighter buoyant density than the calnexin-containing ER
membranes. In addition, the vast majority of the 3A protein in these
transfected cells was also detected in this light fraction. Therefore,
2BC expression was sufficient to recruit the 3A protein quantitatively
into membranes similar in buoyant density to those induced during
poliovirus infection, even though 3A colocalized to ER membranes when
expressed in isolation (Fig. 3) (13).
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The morphology of cells that express 2BC and 3A resembles that of poliovirus infection. Ultrastructural analysis of COS-1 cells transfected with the 2BC and 3A expression plasmids revealed morphologies highly reminiscent of poliovirus infection, with both small clustered vesicles and large clear vacuoles formed (Fig. 6B). Higher magnifications revealed that many of the smaller membranous vesicles formed contained double membranes and cytoplasmic lumen (Fig. 6C), were similar in size and shape to the vesicles induced during poliovirus infection (Fig. 1B and C), and tended to be clustered in the centrosomal regions of the transfected cells. As in poliovirus infection (Fig. 1D), the 2C epitopes were found predominantly on or in the immediate vicinity of the double-membraned vesicles (Fig. 6D). Although empty vacuoles such as those found during expression of 2BC alone (Fig. 5B) were observed, they were not heavily labeled with the anti-2C antibody (Fig. 6D). Therefore, the vesicles induced by coexpression of 3A and 2BC resembled the vesicles induced during poliovirus infection more closely than the vesicles induced by expression of 2BC alone.
3A protein colocalizes with 2C in fractions less dense than ER
during poliovirus infection.
To determine whether, during
poliovirus infection, viral 3A protein relocalized from the ER to
lighter, 2BC-containing fractions as it did during coexpression with
2BC protein, the cytoplasmic organelles from poliovirus-infected COS-1
were separated on 12% stock isotonic Percoll gradients. The
3A-containing fractions completely colocalized with the 2C-containing
fractions at a buoyant density lighter than that of the
calnexin-containing ER fractions in extracts from infected cells
(Fig. 7A). In these fractions, 2C, 3A,
and their precursors 3AB and 2BC were all present (data not shown), as
expected for poliovirus replication complexes (6, 18).
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The buoyant density of both virally induced and ER membranes increases after KCl treatment. To monitor physical attributes of the various membrane organelles other than protein composition, we tested the effect of increased KCl concentration on their fractionation in Percoll gradients. When organelles from poliovirus-infected and uninfected COS-1 cells were separated in the presence and absence of 60 mM KCl, the buoyant densities of Golgi, lysosomes, and mitochondria remained unchanged (Fig. 7C and D). However, both the calnexin-containing ER membranes and the 3A- and 2C-containing membranes showed large increases in buoyant density in the presence of 60 mM KCl (Fig. 7A and B). Thus, the membranes incorporated into the virus-induced vesicles and ER membranes responded similarly to ionic change.
Endosomal and poliovirus-induced membranes do not
cofractionate.
Modified endosomal membranes have been
suggested to support the RNA replication complexes of other
positive-strand RNA viruses such as Semliki Forest virus and rubella
virus (19, 29). The distribution of organelles containing
Rab9, an endosomal marker, was determined for poliovirus-infected
cells. The distribution of Rab9 was disperse, making it
difficult to determine the extent of colocalization, or lack
thereof, with the 2C-containing poliovirus-induced membranes. However, upon the addition of 60 mM KCl to the Percoll gradients, the 2C-containing and Rab9-containing membranes responded quite differently (Fig. 8). Specifically,
the distribution of the Rab9-containing endosomal fractions obtained
from the poliovirus-infected cells was not substantially altered by the
addition of KCl (Fig. 8A), whereas the 2C-containing membranes
displayed a higher apparent buoyant density in the presence of 60 mM
KCl (Fig. 8B). Therefore, although there may be some endosomal
membranes or proteins present in the poliovirus-induced membranes, the
virally induced membranes did not display the osmotic properties of
endosomes and the cellular endosomal population was not consumed during
poliovirus infection.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Poliovirus-induced vesicles derive from the ER by the action of 2BC and 3A proteins. Several hypotheses have been proposed to explain the origin of the large numbers of membranous vesicles that accumulate in poliovirus-infected cells. Ultrastructural evidence for the budding of poliovirus-induced vesicles from ER membranes has been reported (5). The disappearance of the Golgi apparatus in poliovirus-infected cells (8, 10, 12, 41, 48) is consistent with the idea that the virus-induced vesicles derive from dispersed Golgi membranes (39). The inhibition of poliovirus replication by brefeldin A (24, 30), which inhibits retrograde traffic from the Golgi to the ER and causes mixing of the Golgi and ER compartments, argues that either Golgi-to-ER traffic or the integrity of the ER and Golgi are critical in supporting poliovirus infection.
Although the buoyant densities of the poliovirus-induced vesicles were not identical to either ER, Golgi, lysosomes, endosomes, or mitochondria, they were closest to that of ER membranes (Fig. 2 and 8). Upon changes in ionic strength of the density gradient media, the poliovirus-induced vesicles behaved most like ER (Fig. 7). Poliovirus 2C and 3A cofractionated with ER membranes in Percoll gradients when expressed together in the absence of other viral proteins (Fig. 4). In contrast, 3A protein coexpressed with 2BC or in the context of poliovirus infection quantitatively localized to membranes whose buoyant density was lighter than that of ER (Fig. 6 and 8). The simplest explanation of these data is that 2BC localizes to the ER and causes the formation of membranous vesicles into which viral 3A protein is recruited but most resident cellular ER proteins are excluded. Further support for the derivation of the poliovirus-induced vesicles from the ER comes from studies in which host cells exhibited an increase in free cytosolic calcium concentrations as the infectious cycle of poliovirus progressed. The independent expression of 2BC, but not 2C, was sufficient to cause this increase in intracellular calcium concentration (23). The loosely bound pool of calcium utilized in second messenger signaling systems is stored within the ER (reviewed in reference 50), suggesting the possibility that the large increase in cytosolic calcium that occurs during poliovirus infection may result from the disruption of ER membranes by the action of 2BC. It has been reported previously (1, 10, 48) that the expression of poliovirus 2BC protein mimics the morphology of poliovirus-infected cells. Consistent with this idea, the same decrease in buoyant density was observed for membranes isolated from poliovirus-infected and 2BC-transfected cells. Furthermore, we observed in the present study a small number of membranous vesicles similar in morphology to those observed during poliovirus infection. However, most of the membranous vesicles containing 2C epitopes (Fig. 6C) that formed during the expression of 2BC in isolation were much larger than those observed during poliovirus infection. The 2BC-induced membranes further differed from poliovirus-induced vesicles in that they displayed single membranes and lacked electron-dense material in the lumen (Fig. 6B and C). In contrast, by both biochemical and ultrastructural criteria, the membrane rearrangements observed in cells that expressed both 3A and 2BC resembled those observed during poliovirus infection. Membrane vesicles that displayed double membranes, cytoplasmic lumenal contents, and substantial immunolabeling by anti-2C antibody were observed both in poliovirus-infected cells (Fig. 1B and C) (41) and in cells that coexpressed 3A and 2BC (Fig. 6B and C). Furthermore, 3A was recruited into the membranes that contain 2BC protein in both poliovirus-infected (Fig. 7A) and 3A- and 2BC-transfected (Fig. 6A) cells. Whether the interaction between 2BC and 3A proteins is direct or indirect, mediated by cellular proteins or lipids, remains to be determined.Similarities between vesicles induced by positive-strand RNA viruses and autophagic vacuoles. The appearance of double-membraned vesicles is not unique to cells infected with picornaviruses; double-membraned vesicles have been observed in cells infected with arteriviruses (34) and coronaviruses (15) as well. The observation that these virus-induced vesicles are bounded by double lipid bilayers argued that they might form by a mechanism similar to that of autophagic vacuoles (12, 41). Other observations consistent with an autophagy-like origin for the poliovirus-induced vesicles are (i) the apparent ER origin (reference 5 and this work), even though resident ER proteins are, for the most part, excluded (Fig. 2); (ii) the presence of lysosomal enzymes in the vesicles (41), even though they derive from the ER; and (iii) the ability of 2C protein to cause invaginations into ER membranes such that pools of cytoplasm are trapped within ER membranes that contain 3A protein (Fig. 4), providing a potential intermediate in the formation of double-membraned vesicles with cytoplasmic lumen (12, 41). Like the poliovirus-induced vesicles studied here, autophagic vacuoles isolated from primary rat hepatocytes migrate very similarly to ER membranes in Percoll gradients, at a lower buoyant density than lysosomes (32). For both autophagic vacuoles and poliovirus-induced vesicles, the apparent exclusion from the ER of protein markers such as calnexin and PDI could be a selective sorting process.
A model for the formation of poliovirus-induced membranes is shown in Fig. 9. In the presence of both 2BC and 3A, double-membraned vesicles that surround cytoplasm and contain both 2BC and 3A protein but exclude resident ER proteins are formed. The formation of predominately single-membraned vesicles by 2BC in isolation can be rationalized by two models. First, it is possible that in the absence of 3A protein, the engagement of the second membrane that changes a budding mechanism into a wrapping mechanism is not accomplished. A second possibility is that, by analogy to the maturation of autophagic vacuoles, 2BC induces the formation of double-membraned vesicles, but in the absence of 3A protein, they rapidly mature into degradative organelles that have lost their inner membranes and cytoplasmic lumen.
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Formation of viral RNA replication complexes on intact or rearranged intracellular membranes. There is disparity in the literature concerning the cellular origin of the membranes on which positive-strand RNA viruses assemble their RNA replication complexes. If, as we suggest, virus-induced membranes for many positive-strand RNA viruses are formed from the ER by a process that is similar to cellular autophagy, the presence of markers from later in the secretory pathway as well as endosomal contents can be readily explained. For autophagic vacuoles, delivery of extracellular materials such as colloidal gold has been reported to occur within 10 min of its addition to the extracellular milieu (28). Therefore, the presence of these markers does not necessary argue for an endosomal or lysosomal origin for the membranous vesicles induced by positive-strand RNA viruses and is just as consistent with an autophagic origin for the cytopathic vacuoles formed during alphavirus, rubella virus, and mouse hepatitis virus infection (19, 29, 35, 52).
The molecular biology of autophagy in mammalian cells is just beginning to be investigated but may play a significant role in host-pathogen interactions. Twelve genes in Saccharomyces cerevisiae are known to be required for autophagy in yeast (4); it has been demonstrated recently that the human homolog of one of them, beclin-1, is required for the induction of autophagy in human cells (27) and reduces Sindbis virus pathogenesis in mice (26). The maturation of vaccinia virus (44) and African swine fever virus (2), both DNA viruses, involves the addition of two membranes acquired by wrapping subviral particles by the intermediate compartment and the ER, respectively. The pathogenic bacterium Legionella pneumophila grows in modified membranous compartments within infected mammalian cells; these compartments are thought to form by an autophagic mechanism by virtue of their double lipid bilayers derived, at least in part, from the ER (47). Mutant bacteria that fail to thrive in mouse macrophages showed an increase in the accumulation of lysosomal markers and degradative enzymes in these compartments, suggesting that the wild-type bacterial proteins prevent normal maturation of the autophagic vacuoles into degradative compartments (3). Finally, as we have shown here, poliovirus proteins 2BC and 3A, which mimic the biochemical and morphological changes that occur during poliovirus infection, are likely to promote a process similar to cellular autophagy, potentially providing valuable tools to dissect this crucial cellular process.| |
ACKNOWLEDGMENTS |
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We thank our colleagues Peter Sarnow, Amy Clewell, Stephen Deitz, Dana Dodd, Stanley Falkow, Erin Gaynor, Richard Scheller, and Elizabeth Stillman for comments on the manuscript, and we are grateful to John Doedens and Dana Dodd for performing initial transfection experiments. We thank Kurt Bienz, Denise Egger, Gerry Waters, Sue Pierce, Hans-Peter Hauri, and Suzanne Pfeffer for antibodies used in this study.
This work was supported by the National Institutes of Health and by the Hutchison Program for Translational Medicine of Stanford University.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305. Phone: (650) 498-7075. Fax: (650) 498-7147. E-mail: karlak{at}leland.stanford.edu.
Present address: PPD Discovery, Inc., Menlo Park, CA
94025-1435.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Aldabe, R., and L. Carrasco. 1995. Induction of membrane proliferation by poliovirus proteins 2C and 2BC. Biochem. Biophys. Res. Commun. 206:64-76[CrossRef][Medline]. |
| 2. |
Andres, G.,
R. Garcia-Escudero,
C. Simon-Mateo, and E. Vinuela.
1998.
African swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum.
J. Virol.
72:8988-9001 |
| 3. |
Andrews, H. L.,
J. P. Vogel, and R. R. Isberg.
1998.
Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway.
Infect. Immun.
66:950-958 |
| 4. |
Baba, M.,
M. Osumi,
S. V. Scott,
D. J. Klionsky, and Y. Ohsumi.
1997.
Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome.
J. Cell Biol.
139:1687-1695 |
| 5. | Bienz, K., D. Egger, and L. Pasamontes. 1987. Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography. Virology 160:220-226[CrossRef][Medline]. |
| 6. |
Bienz, K.,
D. Egger,
T. Pfjister, and M. Troxler.
1992.
Structural and functional characterization of the poliovirus replication complex.
J. Virol.
66:2740-2747 |
| 7. |
Bienz, K.,
D. Egger,
M. Troxler, and L. Pasamontes.
1990.
Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region.
J. Virol.
64:1156-1163 |
| 8. |
Bienz, K.,
D. Egger, and D. A. Wolff.
1973.
Virus replication, cytopathology, and lysosomal enzyme response of mitotic and interphase HEp-2 cells infected with poliovirus.
J. Virol.
11:565-574 |
| 9. |
Carette, J. E.,
M. Stuiver,
J. van Lent,
J. Wellink, and A. van Kammen.
2000.
Cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not Golgi membranes and is dependent on de novo membrane synthesis.
J. Virol.
74:6556-6563 |
| 10. | Cho, M. W., N. Teterina, D. Egger, K. Bienz, and E. Ehrenfeld. 1994. Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2BC in human cells. Virology 202:129-145[CrossRef][Medline]. |
| 11. | Dahl, R., and L. A. Staehelin. 1989. High-pressure freezing for the preservation of biological structure: theory and practice. J. Electron Microsc. Tech. 13:165-174[CrossRef][Medline]. |
| 12. | Dales, S., H. J. Eggers, I. Tamm, and G. E. Palade. 1965. Electron microscopic study of the formation of poliovirus. Virology 26:379-389[CrossRef][Medline]. |
| 13. | Doedens, J. R., T. H. Giddings, Jr., and K. Kirkegaard. 1997. Inhibition of ER-to-Golgi traffic by poliovirus protein 3A: genetic and ultrastructural analysis. J. Virol. 71:9054-9064[Abstract]. |
| 14. | Doedens, J. R., and K. Kirkegaard. 1995. Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A. EMBO J. 14:894-907[Medline]. |
| 15. | Dubois-Dalcq, M., K. V. Holmes, and B. Rentier. 1984. Assembly of enveloped RNA viruses. Springer-Verlag, Vienna, Austria. |
| 16. |
Dunn, W. A., Jr.
1990.
Studies on the mechanisms of autophagy: formation of the autophagic vacuole.
J. Cell Biol.
110:1923-1933 |
| 17. |
Dunn, W. A., Jr.
1990.
Studies on the mechanisms of autophagy: maturation of the autophagic vacuole.
J. Cell Biol.
110:1935-1945 |
| 18. | Egger, D., L. Pasamontes, R. Bolten, V. Boyko, and K. Bienz. 1996. Reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral RNA synthesis. J. Virol. 70:8675-8683[Abstract]. |
| 19. |
Froshauer, S.,
J. Kartenbeck, and A. Helenius.
1988.
Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes.
J. Cell Biol.
107:2075-2086 |
| 20. | Green, J. M., L. Gu, C. Ifkovits, P. T. Kaumaya, S. Conrad, and S. K. Pierce. 1995. Generation and characterization of monoclonal antibodies specific for members of the mammalian 70-kDa heat shock protein family. Hybridoma 14:347-354[Medline]. |
| 21. |
Guskey, L. E.,
P. C. Smith, and D. A. Wolff.
1970.
Patterns of cytopathology and lysosomal enzyme release in poliovirus-infected HEp-2 cells treated with either 2-(-hydroxybenzyl)-benzimidazole or guanidine HCl.
J. Gen. Virol.
6:151-161 |
| 22. | Hope, D. A., S. E. Diamond, and K. Kirkegaard. 1997. Genetic dissection of interaction between poliovirus RNA-dependent RNA polymerase and viral protein 3AB. J. Virol. 71:9490-9498[Abstract]. |
| 23. | Irurzun, A., J. Arroyo, A. Alvarez, and L. Carrasco. 1995. Enhanced intracellular calcium concentration during poliovirus infection. J. Virol. 69:5142-5146[Abstract]. |
| 24. | Irurzun, A., L. Perez, and L. Carrasco. 1992. Involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin A. Virology 191:166-175[CrossRef][Medline]. |
| 25. |
Lama, J.,
A. V. Paul,
S. Harris, and E. Wimmer.
1994.
Properties of purified recombinant poliovirus protein 3AB as substrate for viral proteinases and as co-factor for RNA polymerase 3Dpol.
J. Biol. Chem.
269:66-70 |
| 26. |
Liang, X. H.,
L. K. Kleeman,
H. H. Jiang,
G. Gordon,
J. E. Goldman,
B. Herman, and B. Levine.
1998.
Protection against fatal Sindbis virus encephalitis by beclin, a novel BCL-2-interacting protein.
J. Virol.
72:8586-8596 |
| 27. | Liang, X. H., S. Jackson, M. Seaman, K. Brown, B. Kempkes, H. Hibshoosh, and B. Levine. 1999. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402:672-676[CrossRef][Medline]. |
| 28. |
Liou, W.,
H. J. Geuze,
M. J. H. Geelen, and J. W. Slot.
1997.
The autophagic and endocytic pathways converge at the nascent autophagic vacuoles.
J. Cell Biol.
136:61-70 |
| 29. | Magliano, D., J. A. Marshall, D. S. Bowden, N. Vardaxis, J. Meanger, and J.-Y. Lee. 1998. Rubella virus replication complexes are virus-modified lysosomes. Virology 240:57-63[CrossRef][Medline]. |
| 30. |
Maynell, L. A.,
K. Kirkegaard, and M. W. Klymkowsky.
1992.
Inhibition of poliovirus RNA synthesis by brefeldin A.
J. Virol.
66:1985-1994 |
| 31. | Mirzayan, C., and E. Wimmer. 1994. Biochemical studies on poliovirus polypeptide 2C: evidence for ATPase activity. Virology 199:176-187[CrossRef][Medline]. |
| 32. |
Niioka, S.,
M. Goto,
T. Ishibashi, and M. Kadowaki.
1998.
Identification of autolysosomes directly associated with proteolysis on the density gradients in isolated rat hepatocytes.
J. Biochem.
124:1086-1093 |
| 33. | Paul, A. V., J. H. van Boom, D. Filippov, and E. Wimmer. 1998. Protein-primed RNA synthesis by purified poliovirus RNA polymerase. Nature 393:280-284[CrossRef][Medline]. |
| 34. |
Pedersen, I. W.,
Y. van der Meer,
N. Roos, and E. J. Snijder.
1999.
ORF1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double membrane vesicles which carry the viral replication complex.
J. Virol.
73:2016-2026 |
| 35. | Peranen, J., P. Laakkonen, M. Hyvonin, and L. Kaarianen. 1995. The alphavirus replicase protein nsP1 is membrane-associated and has affinity to endocytic organelles. Virology 208:610-620[CrossRef][Medline]. |
| 36. | Peters, T. J., M. Muller, and C. De Duve. 1972. Lysosomes of the arterial wall. I. Isolation and subcellular fractionation of cells from normal rabbit aorta. J. Exp. Med. 136:1117-1139[Abstract]. |
| 37. | Reunanen, H., E. L. Punnonen, and P. Hirsimaki. 1985. Studies on vinblastine-induced autophagocytosis in mouse liver. V. A cytochemical study on the origin of membranes. Histochemistry 83:513-517[CrossRef][Medline]. |
| 38. |
Rodriguez, P. L., and L. Carrasco.
1993.
Poliovirus protein 2C has ATPase and GTPase activities.
J. Biol. Chem.
268:8105-8110 |
| 39. | Sandoval, I. V., and L. Carrasco. 1997. Poliovirus infection and expression of the poliovirus protein 2B provoke the disassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179. J. Virol. 71:4679-4693[Abstract]. |
| 40. | Schagger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368-379[CrossRef][Medline]. |
| 41. |
Schlegel, A.,
T. H. Giddings,
M. S. Ladinsky, and K. Kirkegaard.
1996.
Cellular origin and ultrastructure of membranes induced during poliovirus infection.
J. Virol.
70:6576-6588 |
| 42. | Schweizer, A., J. Rohrer, J. W. Slot, H. J. Geuze, and S. Kornfeld. 1995. Reassessment of the subcellular localization of p63. J. Cell Sci. 108:2477-2485[Abstract]. |
| 43. | Sheeler, P. 1981. Density gradient centrifugation, p. 81-99. In P. Sheeler (ed.), Centrifugation in biology and medical science. John Wiley & Sons, Inc., New York, N.Y. |
| 44. |
Sodiek, B.,
R. W. Doms,
M. Ericsson,
G. Hiller,
C. E. Machamer,
W. van't Hof,
G. van Meer,
B. Moss, and G. Griffiths.
1993.
Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks.
J. Cell Biol.
121:521-541 |
| 45. | Stromhaug, P. E., T. O. Berg, M. Fengsrud, and P. O. Seglen. 1998. Purification and characterization of autophagosomes from rat hepatocytes. Biochem. J. 335:217-224. |
| 46. |
Suhy, D. A.,
K. D. Simon,
D. I. Linzer, and T. V. O'Halloran.
1999.
Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivation.
J. Biol. Chem.
274:9183-9192 |
| 47. | Swanson, M. S., and R. R. Isberg. 1995. Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect. Immun. 63:3609-3620[Abstract]. |
| 48. | Teterina, N. L., A. E. Gorbalenya, D. Egger, K. Bienz, and E. Ehrenfeld. 1997. Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells. J. Virol. 71:8962-8972[Abstract]. |
| 49. |
Towner, J., and B. L. Semler.
1996.
Determinants of membrane association on poliovirus protein 3AB.
J. Biol. Chem.
271:26810-26818 |
| 50. | Tsien, R. W., and R. Y. Tsien. 1990. Calcium channels, stores and oscillations. Annu. Rev. Cell Biol. 6:715-760[CrossRef]. |
| 51. |
Ueno, T.,
K. Ishidoh,
R. Mineki,
I. Tanida,
K. Murayama,
M. Kadowaki, and E. Kominami.
1999.
Autolysosomal membrane-associated betaine homocysteine methyltransferase. Limited degradation fragment of a sequestered cytosolic enzyme monitoring autophagy.
J. Biol. Chem.
274:15222-15229 |
| 52. |
van der Meer, Y.,
E. J. Snijder,
J. C. Dobbe,
S. Schleich,
M. R. Denison,
W. J. M. Spaan, and J. Krijnse Locker.
1999.
Location of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication.
J. Virol.
73:7641-7657 |
| 53. |
Waters, M. G.,
D. O. Clary, and J. E. Rothman.
1992.
A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack.
J. Cell Biol.
118:1015-1026 |
| 54. | Watzele, G., R. Bachofner, and E. G. Berger. 1991. Immunocytochemical localization of the Golgi apparatus using protein-specific antibodies to galactosyltransferase. Eur. J. Cell Biol. 56:451-458[Medline]. |
| 55. | Wessel, D., and U. I. Flugge. 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138:141-143[CrossRef][Medline]. |
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80: 5927-5940
[Abstract] [Full Text] -
Berkova, Z., Crawford, S. E., Trugnan, G., Yoshimori, T., Morris, A. P., Estes, M. K.
(2006). Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms.. J. Virol.
80: 6061-6071
[Abstract] [Full Text] -
de Jong, A. S., Visch, H.-J., de Mattia, F., van Dommelen, M. M., Swarts, H. G., Luyten, T., Callewaert, G., Melchers, W. J., Willems, P. H., van Kuppeveld, F. J.
(2006). The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi. J. Biol. Chem.
281: 14144-14150
[Abstract] [Full Text] -
Wileman, T.
(2006). Aggresomes and autophagy generate sites for virus replication.. Science
312: 875-878
[Abstract] [Full Text] -
Lerner, R. S., Nicchitta, C. V.
(2006). mRNA translation is compartmentalized to the endoplasmic reticulum following physiological inhibition of cap-dependent translation. RNA
12: 775-789
[Abstract] [Full Text] -
Nair, U., Klionsky, D. J.
(2005). Molecular Mechanisms and Regulation of Specific and Nonspecific Autophagy Pathways in Yeast. J. Biol. Chem.
280: 41785-41788
[Full Text] -
Barretto, N., Jukneliene, D., Ratia, K., Chen, Z., Mesecar, A. D., Baker, S. C.
(2005). The Papain-Like Protease of Severe Acute Respiratory Syndrome Coronavirus Has Deubiquitinating Activity. J. Virol.
79: 15189-15198
[Abstract] [Full Text] -
Zhang, S. C., Zhang, G., Yang, L., Chisholm, J., Sanfacon, H.
(2005). Evidence that Insertion of Tomato Ringspot Nepovirus NTB-VPg Protein in Endoplasmic Reticulum Membranes Is Directed by Two Domains: a C-Terminal Transmembrane Helix and an N-Terminal Amphipathic Helix. J. Virol.
79: 11752-11765
[Abstract] [Full Text] -
Belov, G. A., Fogg, M. H., Ehrenfeld, E.
(2005). Poliovirus Proteins Induce Membrane Association of GTPase ADP-Ribosylation Factor. J. Virol.
79: 7207-7216
[Abstract] [Full Text] -
Harris, J. R., Racaniello, V. R.
(2005). Amino Acid Changes in Proteins 2B and 3A Mediate Rhinovirus Type 39 Growth in Mouse Cells. J. Virol.
79: 5363-5373
[Abstract] [Full Text] -
Wessels, E., Duijsings, D., Notebaart, R. A., Melchers, W. J. G., van Kuppeveld, F. J. M.
(2005). A Proline-Rich Region in the Coxsackievirus 3A Protein Is Required for the Protein To Inhibit Endoplasmic Reticulum-to-Golgi Transport. J. Virol.
79: 5163-5173
[Abstract] [Full Text] -
Moffat, K., Howell, G., Knox, C., Belsham, G. J., Monaghan, P., Ryan, M. D., Wileman, T.
(2005). Effects of Foot-and-Mouth Disease Virus Nonstructural Proteins on the Structure and Function of the Early Secretory Pathway: 2BC but Not 3A Blocks Endoplasmic Reticulum-to-Golgi Transport. J. Virol.
79: 4382-4395
[Abstract] [Full Text] -
Knox, C., Moffat, K., Ali, S., Ryan, M., Wileman, T.
(2005). Foot-and-mouth disease virus replication sites form next to the nucleus and close to the Golgi apparatus, but exclude marker proteins associated with host membrane compartments. J. Gen. Virol.
86: 687-696
[Abstract] [Full Text] -
Egger, D., Bienz, K.
(2005). Intracellular location and translocation of silent and active poliovirus replication complexes. J. Gen. Virol.
86: 707-718
[Abstract] [Full Text] -
Klionsky, D. J.
(2005). The molecular machinery of autophagy: unanswered questions. J. Cell Sci.
118: 7-18
[Abstract] [Full Text] -
Shintani, T., Klionsky, D. J.
(2004). Autophagy in Health and Disease: A Double-Edged Sword. Science
306: 990-995
[Abstract] [Full Text] -
Elazar, M., Liu, P., Rice, C. M., Glenn, J. S.
(2004). An N-Terminal Amphipathic Helix in Hepatitis C Virus (HCV) NS4B Mediates Membrane Association, Correct Localization of Replication Complex Proteins, and HCV RNA Replication. J. Virol.
78: 11393-11400
[Abstract] [Full Text] -
Banerjee, R., Weidman, M. K., Echeverri, A., Kundu, P., Dasgupta, A.
(2004). Regulation of Poliovirus 3C Protease by the 2C Polypeptide. J. Virol.
78: 9243-9256
[Abstract] [Full Text] -
Schwartz, M., Chen, J., Lee, W.-M., Janda, M., Ahlquist, P.
(2004). Alternate, virus-induced membrane rearrangements support positive-strand RNA virus genome replication. Proc. Natl. Acad. Sci. USA
101: 11263-11268
[Abstract] [Full Text] -
Choe, S. S., Kirkegaard, K.
(2004). Intracellular Topology and Epitope Shielding of Poliovirus 3A Protein. J. Virol.
78: 5973-5982
[Abstract] [Full Text] -
de Jong, A. S., Melchers, W. J. G., Glaudemans, D. H. R. F., Willems, P. H. G. M., van Kuppeveld, F. J. M.
(2004). Mutational Analysis of Different Regions in the Coxsackievirus 2B Protein: REQUIREMENTS FOR HOMO-MULTIMERIZATION, MEMBRANE PERMEABILIZATION, SUBCELLULAR LOCALIZATION, AND VIRUS REPLICATION. J. Biol. Chem.
279: 19924-19935
[Abstract] [Full Text] -
Grubman, M. J., Baxt, B.
(2004). Foot-and-Mouth Disease. Clin. Microbiol. Rev.
17: 465-493
[Abstract] [Full Text] -
Monaghan, P., Cook, H., Jackson, T., Ryan, M., Wileman, T.
(2004). The ultrastructure of the developing replication site in foot-and-mouth disease virus-infected BHK-38 cells. J. Gen. Virol.
85: 933-946
[Abstract] [Full Text] -
Crotty, S., Saleh, M.-C., Gitlin, L., Beske, O., Andino, R.
(2004). The Poliovirus Replication Machinery Can Escape Inhibition by an Antiviral Drug That Targets a Host Cell Protein. J. Virol.
78: 3378-3386
[Abstract] [Full Text] -
Prentice, E., Jerome, W. G., Yoshimori, T., Mizushima, N., Denison, M. R.
(2004). Coronavirus Replication Complex Formation Utilizes Components of Cellular Autophagy. J. Biol. Chem.
279: 10136-10141
[Abstract] [Full Text] -
Wang, A., Han, S., Sanfacon, H.
(2004). Topogenesis in membranes of the NTB-VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain. J. Gen. Virol.
85: 535-545
[Abstract] [Full Text] -
Miyanari, Y., Hijikata, M., Yamaji, M., Hosaka, M., Takahashi, H., Shimotohno, K.
(2003). Hepatitis C Virus Non-structural Proteins in the Probable Membranous Compartment Function in Viral Genome Replication. J. Biol. Chem.
278: 50301-50308
[Abstract] [Full Text] -
Teterina, N. L., Rinaudo, M. S., Ehrenfeld, E.
(2003). Strand-Specific RNA Synthesis Defects in a Poliovirus with a Mutation in Protein 3A. J. Virol.
77: 12679-12691
[Abstract] [Full Text] -
Lee, W.-M., Ahlquist, P.
(2003). Membrane Synthesis, Specific Lipid Requirements, and Localized Lipid Composition Changes Associated with a Positive-Strand RNA Virus RNA Replication Protein. J. Virol.
77: 12819-12828
[Abstract] [Full Text] -
Fogg, M. H., Teterina, N. L., Ehrenfeld, E.
(2003). Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro. J. Virol.
77: 11408-11416
[Abstract] [Full Text] -
Ettayebi, K., Hardy, M. E.
(2003). Norwalk Virus Nonstructural Protein p48 Forms a Complex with the SNARE Regulator VAP-A and Prevents Cell Surface Expression of Vesicular Stomatitis Virus G Protein. J. Virol.
77: 11790-11797
[Abstract] [Full Text] -
El-Hage, N., Luo, G.
(2003). Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA. J. Gen. Virol.
84: 2761-2769
[Abstract] [Full Text] -
Krogerus, C., Egger, D., Samuilova, O., Hyypia, T., Bienz, K.
(2003). Replication Complex of Human Parechovirus 1. J. Virol.
77: 8512-8523
[Abstract] [Full Text] -
Konan, K. V., Giddings, T. H. Jr., Ikeda, M., Li, K., Lemon, S. M., Kirkegaard, K.
(2003). Nonstructural Protein Precursor NS4A/B from Hepatitis C Virus Alters Function and Ultrastructure of Host Secretory Apparatus. J. Virol.
77: 7843-7855
[Abstract] [Full Text] -
Pattingre, S., De Vries, L., Bauvy, C., Chantret, I., Cluzeaud, F., Ogier-Denis, E., Vandewalle, A., Codogno, P.
(2003). The G-protein Regulator AGS3 Controls an Early Event during Macroautophagy in Human Intestinal HT-29 Cells. J. Biol. Chem.
278: 20995-21002
[Abstract] [Full Text] -
Elazar, M., Cheong, K. H., Liu, P., Greenberg, H. B., Rice, C. M., Glenn, J. S.
(2003). Amphipathic Helix-Dependent Localization of NS5A Mediates Hepatitis C Virus RNA Replication. J. Virol.
77: 6055-6061
[Abstract] [Full Text] -
Harris, J. R., Racaniello, V. R.
(2003). Changes in Rhinovirus Protein 2C Allow Efficient Replication in Mouse Cells. J. Virol.
77: 4773-4780
[Abstract] [Full Text] -
Salonen, A., Vasiljeva, L., Merits, A., Magden, J., Jokitalo, E., Kaariainen, L.
(2003). Properly Folded Nonstructural Polyprotein Directs the Semliki Forest Virus Replication Complex to the Endosomal Compartment. J. Virol.
77: 1691-1702
[Abstract] [Full Text] -
de Jong, A. S., Wessels, E., Dijkman, H. B. P. M., Galama, J. M. D., Melchers, W. J. G., Willems, P. H. G. M., van Kuppeveld, F. J. M.
(2003). Determinants for Membrane Association and Permeabilization of the Coxsackievirus 2B Protein and the Identification of the Golgi Complex as the Target Organelle. J. Biol. Chem.
278: 1012-1021
[Abstract] [Full Text] -
Jurgens, C., Flanegan, J. B.
(2002). Initiation of Poliovirus Negative-Strand RNA Synthesis Requires Precursor Forms of P2 Proteins. J. Virol.
77: 1075-1083
[Abstract] [Full Text] -
Han, S., Sanfacon, H.
(2002). Tomato Ringspot Virus Proteins Containing the Nucleoside Triphosphate Binding Domain Are Transmembrane Proteins That Associate with the Endoplasmic Reticulum and Cofractionate with Replication Complexes. J. Virol.
77: 523-534
[Abstract] [Full Text] -
Egger, D., Bienz, K.
(2002). Recombination of Poliovirus RNA Proceeds in Mixed Replication Complexes Originating from Distinct Replication Start Sites. J. Virol.
76: 10960-10971
[Abstract] [Full Text] -
Gazina, E. V., Mackenzie, J. M., Gorrell, R. J., Anderson, D. A.
(2002). Differential Requirements for COPI Coats in Formation of Replication Complexes among Three Genera of Picornaviridae. J. Virol.
76: 11113-11122
[Abstract] [Full Text] -
Linardakis, E., Bateman, A., Phan, V., Ahmed, A., Gough, M., Olivier, K., Kennedy, R., Errington, F., Harrington, K. J., Melcher, A., Vile, R.
(2002). Enhancing the Efficacy of a Weak Allogeneic Melanoma Vaccine by Viral Fusogenic Membrane Glycoprotein-mediated Tumor Cell-Tumor Cell Fusion. Cancer Res.
62: 5495-5504
[Abstract] [Full Text] -
Green, K. Y., Mory, A., Fogg, M. H., Weisberg, A., Belliot, G., Wagner, M., Mitra, T., Ehrenfeld, E., Cameron, C. E., Sosnovtsev, S. V.
(2002). Isolation of Enzymatically Active Replication Complexes from Feline Calicivirus-Infected Cells. J. Virol.
76: 8582-8595
[Abstract] [Full Text] -
Ritzenthaler, C., Laporte, C., Gaire, F., Dunoyer, P., Schmitt, C., Duval, S., Piequet, A., Loudes, A. M., Rohfritsch, O., Stussi-Garaud, C., Pfeiffer, P.
(2002). Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes. J. Virol.
76: 8808-8819
[Abstract] [Full Text] -
Lyle, J. M., Bullitt, E., Bienz, K., Kirkegaard, K.
(2002). Visualization and Functional Analysis of RNA-Dependent RNA Polymerase Lattices. Science
296: 2218-2222
[Abstract] [Full Text] -
Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D., Bienz, K.
(2002). Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex. J. Virol.
76: 5974-5984
[Abstract] [Full Text] -
Carette, J. E., Guhl, K., Wellink, J., Van Kammen, A.
(2002). Coalescence of the Sites of Cowpea Mosaic Virus RNA Replication into a Cytopathic Structure. J. Virol.
76: 6235-6243
[Abstract] [Full Text] -
Carette, J. E., van Lent, J., MacFarlane, S. A., Wellink, J., van Kammen, A.
(2002). Cowpea Mosaic Virus 32- and 60-Kilodalton Replication Proteins Target and Change the Morphology of Endoplasmic Reticulum Membranes. J. Virol.
76: 6293-6301
[Abstract] [Full Text] -
de Jong, A. S., Schrama, I. W. J., Willems, P. H. G. M., Galama, J. M. D., Melchers, W. J. G., van Kuppeveld, F. J. M.
(2002). Multimerization reactions of coxsackievirus proteins 2B, 2C and 2BC: a mammalian two-hybrid analysis. J. Gen. Virol.
83: 783-793
[Abstract] [Full Text] -
Brass, V., Bieck, E., Montserret, R., Wolk, B., Hellings, J. A., Blum, H. E., Penin, F., Moradpour, D.
(2002). An Amino-terminal Amphipathic alpha -Helix Mediates Membrane Association of the Hepatitis C Virus Nonstructural Protein 5A. J. Biol. Chem.
277: 8130-8139
[Abstract] [Full Text] -
den Boon, J. A., Chen, J., Ahlquist, P.
(2001). Identification of Sequences in Brome Mosaic Virus Replicase Protein 1a That Mediate Association with Endoplasmic Reticulum Membranes. J. Virol.
75: 12370-12381
[Abstract] [Full Text] -
Schmidt-Mende, J., Bieck, E., Hugle, T., Penin, F., Rice, C. M., Blum, H. E., Moradpour, D.
(2001). Determinants for Membrane Association of the Hepatitis C Virus RNA-dependent RNA Polymerase. J. Biol. Chem.
276: 44052-44063
[Abstract] [Full Text] -
Rust, R. C., Landmann, L., Gosert, R., Tang, B. L., Hong, W., Hauri, H.-P., Egger, D., Bienz, K.
(2001). Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex. J. Virol.
75: 9808-9818
[Abstract] [Full Text] -
Dodd, D. A., Giddings, T. H. Jr., Kirkegaard, K.
(2001). Poliovirus 3A Protein Limits Interleukin-6 (IL-6), IL-8, and Beta Interferon Secretion during Viral Infection. J. Virol.
75: 8158-8165
[Abstract] [Full Text] -
Deitz, S. B., Dodd, D. A., Cooper, S., Parham, P., Kirkegaard, K.
(2000). MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A. Proc. Natl. Acad. Sci. USA
10.1073/pnas.250483097v1
[Abstract] [Full Text] -
Talloczy, Z., Jiang, W., Virgin, H. W. IV, Leib, D. A., Scheuner, D., Kaufman, R. J., Eskelinen, E.-L., Levine, B.
(2002). Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway. Proc. Natl. Acad. Sci. USA
99: 190-195
[Abstract] [Full Text] -
Deitz, S. B., Dodd, D. A., Cooper, S., Parham, P., Kirkegaard, K.
(2000). MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A. Proc. Natl. Acad. Sci. USA
97: 13790-13795
[Abstract] [Full Text]
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